Purification and Characterization of a Glucose tolerant β-Glucosidase from a Newly Isolated Neofusicoccum Parvum strain F7: Production Optimization using Plackett Burman and Box Behnken

Abstract

Abstract Endoglucanases, exoglucanases and β-glucosidases act synergistically to hydrolyse cellulose into glucose monomers. Thus, this study aimed to improve production of a β-glucosidase from a newly isolated Neofusicoccum parvum strain F7 by optimizing the culture conditions and medium components using Plackett-Burman Design (PBD) and Box Behnken Design (BBD). β-Glucosidase production was significantly enhanced (p-value≤0.05) by 1.5-fold to 2.5 U/ml by BBD as compared to the preliminary one variable at a time (OVAT) experiments of (1.6 U/ml). The optimal conditions for enzyme production by BBD were 12 days of fermentation at 20°C, 175 rpm, 0.5% glycerol and 1.5% casein in 50 mM sodium phosphate (pH 6.0) buffer. Three β-glucosidase isoforms referred to as Bgl1, Bgl2, Bgl3 were purified and characterized from the optimized crude extract displaying IC50 values of 2.6, 22.6 and 319.5 mM for glucose, respectively. Bgl3 with a molecular weight of approximately 65 kDa displayed the highest tolerance to glucose among the isoforms. The optimum activity and stability for Bgl3 was observed at pH 4.0 in 50 mM sodium acetate buffer with 80% β-glucosidase residual activity retained for three hours. This isoform also retained 60% residual activity at 65°C for one hour which was then reduced to 40 % which remained stable for another 90 minutes. The β-Glucosidase activity of Bgl3 was not enhanced after the addition of metal ions in assay buffers. The Km and vmax for 4-nitrophenyl-β-D-glucopyranoside were found to be 1.18 mM and 28.08 µmol/min, respectively indicating high affinity to the substrate. The ability to withstand the presence of glucose in conjunction with its thermophilic nature indicates promise for the enzyme in industrial application.